Immunometric assays (also called "sandwich assays") using specific antibodies for the determination of the presence and/or concentration of antigenic substances in fluids are well known.
In such an assay, the amount of labeled antibody associated with a complex is directly proportional to the amount of antigenic substance in the fluid sample, illustrated as follows: ##STR1## This assay which is a "two site" or "sandwich" assay because two non-interfering epitopic sites of the antigen are bound, is described by Wide, "Radioimmunoassay Methods", edited by Kirkham and Hunter, E. & S. Livingston, Edinburgh, 1970, pp. 199-206. Such immunometric assays have been found to be effective for the detection of polyvalent antigen or antibody, i.e., an antigenic substance that is able to complex with two or more antibodies at the same time.
In an attempt to alleviate problems present in the prior art, a method is described by David et al., in U.S. Pat. No. 4,376,110, wherein the polyclonal antibody used in an immunometric assay is replaced by at least one and usually two different monoclonal antibodies, each specific for a single, unique epitope or antigenic site. According to the conventional method, the epitope-specificity of the monoclonal antibody to be coated to the solid support should be different from the monoclonal antibody used for the labeled antibody. The two monoclonal antibodies should bind the antigenic substance at sites remote from each other so that the binding of one antibody will not interfere with the binding of the other.
Many problems have been encountered by reasearchers using the above technique when monoclonal antibodies produced by one or two cell lines are used in the two site sandwich immunometric assay. The assay has suffered from several problems:
1. Lack of low end sensitivity.
At the end of the standard range, the assays may not detect very low levels of analyte. This is particularly true for hepatitis B surface (HB.sub.s) antigen and glycopituitary hormones such as human chorionic gonadotropin (hCG), thyroid stimulating hormone (TSH), luteinizing hormone (LH), and follicle stimulating hormone (FSH). This shortcoming leads, for example, to inability to detect a recent hepatitis infection, or to inability to distinguish normal thyroid function from hyperthyroid function, or conceivably may result in a failure to detect a dangerous ectopic pregnancy.
2. Lack of Accuracy.
Interference occurs in TSH assays run on pregnant patient serum. The structure of one of the subunits of hCG is very similar to a TSH subunit. If present in the serum, the hCG will bind to the monoclonal antibody on the solid carrier and prevent the binding of TSH, which is normally found in very low levels. The labeled monoclonal antibody in solution will not bind to the hCG to complete the "sandwich," so the result is negative, regardless of the actual TSH value.
In an hCG assay, only the intact hCG beta molecule will be recognized by both the solid carrier monoclonal antibody and the labeled monoclonal to complete the "sandwich". However, beta subunits may be found in the serum in cases of hydatidiform mole, choriocarcinoma and any other malignant melanoma and will not be detected by this assay.
3. Hook effect (or prozone phenomenon).
It is well-known that concentration of hCG in pregnancy often exceed 100,000 mIU/ml. Therefore, an assay must give reliable results at high concentrations of hCG. However, a sandwich assay using one monoclonal antibody that is labeled and one on the solid carrier may suffer from a hook effect. The hook effect occurs when high levels of hCG saturate the monoclonal antibody binding sites, disallowing the completion of the sandwich, and "hooking" the results down to a lower level. A very high hCG sample may actually test as negative.
4. Interference.
The presence of non-analyte antibody-binding substances in patient serum has been found by many laboratories to cause a false increase in analyte concentrations when sandwich assays are used. These binding substances may be antibodies which mimic the analyte by binding both the antibody on the solid carrier and the soluble labeled antibody.
This cross-reaction may occur with monoclonal antibodies or with other antisera when antibodies from the same species are used for both the solid phase and the soluble labled phase. The occurence of antibody directed cross-reactivity against monoclonal antibody sandwich assays has been reported for TSH, hCG, carcinoembryonic antigen (CEA), hepatitis A and hepatitis B, prolactin, LH and FSH.
5. Complicated reaction devices
The prior art has required trained technical personnel with specialized laboratory equipment to perform immunoassays. Small and accurately dispensed amounts of reactants had to be reproduceably aliquoted and quantitation required sophisticated spectrophotometric detection instrumentation.
6. Instability of reagents
Refrigerated storage of components was necessary to extend the shelf life of reagents beyond a few weeks. For example, in case of the enzyme substrate 5'-bromo-4-chloro-3-indoxylphosphate (BCIP), the substrate requires a color enhancer such as Nitro-Blue tetrazolium chloride (NBT) for optimum color development. The chemical combination of these two ingredients results in a thermally labile mixture.
7. Extra steps
Previous applications required an additional wash step prior to the addition of the substrate to remove the excess antibody-enzyme conjugate before reaction with the enzyme substrate.
Examples of conventional "sandwich" immunoassays for various antigens such as alpha fetoprotein (AFP), hCG, TSH, LH, FSH, IgE, and hepatitis B-surface antigen are of the following types:
1. Two site immunoassay based on two polyclonal antibodies, one of these antibodies being immobilized on a solid phase and the other being in soluble labeled form.
2. Two site immunoassay based on two monoclonal antibodies directed against two separate and distinct epitopes of the antigen, one of these antibodies being bound on a solid phase and the other being of soluble labeled form (David et al., supra).
3. Two site immunoassay based on one monoclonal antibody and one polyclonal antibody, the monoclonal antibody being bound on a solid phase and the polyclonal antibody being in soluble labeled form.
In view of the mentioned shortcomings, only trained personnel in well equipped clinical laboratories can perform traditional sandwich enzyme immunoassays. Even qualitative assays can result in equivocal answers because of potential misinterpretation of the gradient of color reactions near the cutoff concentration. Thermal instability of commonly employed reagent combinations require refrigerated storage, and incomplete "washing" can yield high background and spurious results.
The conventional forward or reverse assay using the monoclonal-monoclonal antibody sandwich assay system (David et al., supra) is time consuming, and is not well suited to determination of small concentrations of antigen since formation of a sandwich of the antigen involves only two matching epitopes of the monoclonal antibody. The occurrence of false positive (false elevated) or false negative (false lower) values can be due to the nature of the monoclonal-monoclonal paired antibodies and to the cross reactivity of non-related antigen or antigen similar substances.
The conventional monoclonal-monoclonal antibody sandwich simultaneous assay (as opposed to the forward or reverse assay) also has the potential for a hook effect since the number of soluble labeled antibody molecules is limited.
Accordingly, one object of the present invention is to provide an improved process for the sandwich or two-site immunometric assay for antigenic substances.
An object of the present invention is to provide more rapid, more specific and more accurate immunometric assay techniques.
Another object of the present invention is to provide more sensitive immunometric assay techniques.
Another object of the present invention is to provide improved "simultaneous" immunometric assays.
Another object of the invention is to create a simple reaction device for immunometric assays which can be utilized without special equipment or training.
Another object of the invention is to create an enhanced color reaction in a thermally stable reagent configuration.
Another object of the invention is to present the end point of the reaction in an easily interpreted manner with virtually no potential for equivocal results.
Another object of the invention is to eliminate the washing step for membrane based immunoassay procedures.
The manner in which these and other objects are realized by the present invention will be apparent from the summary and detailed description set forth below.